Superparamagnetic Nanobeads Research Articles

Optimizing the functionalization of super-paramagnetic nanobeads for droplet-based protein secretion profiling of immune cells

The extremely dynamic functions of an active immune response are mainly determined by the presence of various soluble factors. The secreted factors organize the individual immune cells into functional tissues regulating the immune response. Dynamical single-cell studies are therefore fundamental to investigate the heterogeneity of immune cells’ behaviors over time. The available tools lack either of single cell resolution or time resolution; new tools are required to access both single cell and time resolution in order to better understand these dynamical single cell mechanisms. The powerful resolution and sensitivity improvement provided by droplet-based microfluidics deciphering the dynamic processes at the single-cell level could help to describe the fundamental mechanisms underlying immunity, develop new strategies for vaccination and cancer immunotherapy, or diagnose inflammatory diseases.The use of functionalized paramagnetic nanoparticles is a key feature of our droplet-based immunoassay. This work describes the optimization and comparison of various functionalization methods for the integration of ready-to-use super-paramagnetic nanobeads into our droplet-based assay. Among the three functionalization tested methods, the most promising one is based on boronic acid chemistry, in terms of both compatibility with the droplet microfluidic system and detection sensitivity. It was successfully applied to the characterization of TNFα secretion from human single monocytes.

Nanomaterial Endocytosis: Quantification of Adsorption and Ingestion Mechanisms

The widespread use of nanomaterials in vaccines, therapeutics, and industrial applications creates an increasing demand for understanding their ingestion by living cells. Researchers in the field have called for a more robust understanding of physical/chemical particle–cell interactions and a means to determine the particles ingested per cell. Using superparamagnetic nanobeads, we measured the beads per cell and quantified the kinetics of the receptor-independent endocytosis of particles having seven surface chemistries. Poly(ethylene glycol) (PEG)-coated nanoparticles were ingested less effectively by cultured Chinese hamster ovary (CHO-K1) cells and more effectively by aminated nanoparticles than starch-coated particles. The cells ingested 2 to 4 × 105 of the most attractive particles. The interplay between Van der Waals and coulombic potentials was quantified on the basis of Derjaguin–Landau–Verwey–Overbeek (DLVO) theory modified to include hydration repulsion using physical parameters of the seven surface chemistries. Using dose–response curves for inhibitors of clathrin- or caveolae-dependent ingestion, we quantified how particle surface chemistry determines which endocytic pathway is used by the cell. Such characterization can be useful in predicting nanomaterial uptake in medical and toxicological applications and in the selection of particle surface chemistries for receptor-dependent endocytosis.

Electrodeposited magnetic nanoporous membrane for high-yield and high-throughput immunocapture of extracellular vesicles and lipoproteins.

Superparamagnetic nanobeads offer several advantages over microbeads for immunocapture of nanocarriers (extracellular vesicles, lipoproteins, and viruses) in a bioassay: high-yield capture, reduction in incubation time, and higher capture capacity. However, nanobeads are difficult to "pull-down" because their superparamagnetic feature requires high nanoscale magnetic field gradients. Here, an electrodeposited track-etched membrane is shown to produce a unique superparamagnetic nano-edge ring with multiple edges around nanopores. With a uniform external magnetic field, the induced monopole and dipole of this nano edge junction combine to produce a 10× higher nanobead trapping force. A dense nanobead suspension can be filtered through the magnetic nanoporous membrane (MNM) at high throughput with a 99% bead capture rate. The yield of specific nanocarriers in heterogeneous media by nanobeads/MNM exceeds 80%. Reproducibility, low loss, and concentration-independent capture rates are also demonstrated. This MNM material hence expands the application of nanobead immunocapture to physiological samples.

Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus

Single-domain antibody (sdAb) or nanobody possesses specific features non-accessible for conventional antibodies that make them suitable for research and biotechnological applications. Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets, resulting in great economic losses all over the world. To detect and isolate PEDV rapidly and accurately is important for the control and further research of the clinical PEDV strains. In this study, four sdAb fragments (sdAb-Mc19/29/30/37) targeting the membrane (M) protein of PEDV were selected from sdAb library that was constructed through M protein-immunized Camelus bactrianus. The selected sdAb-Mcs were solubly expressed in Escherichia coli. The functional characteristics analysis revealed that the recombinant sdAb-Mcs have excellent binding activity and specificity to M protein but have no neutralizing activity to PEDV. For further application, sdAb-Mc37 was conjugated with quantum dots to synthesize a nanoprobe for imaging PEDV in vero cells. The observed fluorescence in vero cells clearly reflects that PEDV virions can be reliably recognized and labeled by the nanoprobe. Furthermore, the sdAb-Mc29 was conjugated with superparamagnetic nanobeads to construct immunomagnetic nanobeads (IMNBs) used to isolate PEDV. One PEDV strain was successfully isolated from clinical fecal sample, suggesting IMNBs as a novel and efficient tool suitable for PEDV isolation from clinical samples. This study provided a novel application and substantiated the suitability of sdAb as a specific binder for the isolation of viruses.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-9324-7) contains supplementary material, which is available to authorized users.

A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads

Lysozyme (Lys) commonly presents in wines and are known to cause toxicological impact on human health. The need of highly sensitive and reliable detection methods are evident in such matrix. In this work, we developed a competitive aptamer based assay for detection of Lys by employing carboxylated magnetic beads as a support to immobilize the target molecule Lys. The used aptamer sequence was biotinylated which further binds with Streptavidin-Alkaline phosphatase (Stp-ALP) in the micro wells. Colorimetric tests were performed in order to optimize different experimental parameters. The Lys assay showed a good linearity in the range of 5–140nM with a limit of detection (LOD) 10nM. The mid-point value (IC50) 110nM and the analysis time (60min) validated the developed aptasensor as a promising tool for routine use. The assay displayed good recoveries of Lys in the range 99.00–99.27% and was demonstrated for the detection of Lys in wine samples.

Rapid detection and quantification of the marine toxic algae, Alexandrium minutum, using a super-paramagnetic immunochromatographic strip test

The dinoflagellates of Alexandrium genus are known to be producers of paralytic shellfish toxins that regularly impact the shellfish aquaculture industry and fisheries. Accurate detection of Alexandrium including Alexandrium minutum is crucial for environmental monitoring and sanitary issues. In this study, we firstly developed a quantitative lateral flow immunoassay (LFIA) using super-paramagnetic nanobeads for A. minutum whole cells. This dipstick assay relies on two distinct monoclonal antibodies used in a sandwich format and directed against surface antigens of this organism. No sample preparation is required. Either frozen or live cells can be detected and quantified. The specificity and sensitivity are assessed by using phytoplankton culture and field samples spiked with a known amount of cultured A. minutum cells. This LFIA is shown to be highly specific for A. minutum and able to detect reproducibly 105cells/L within 30min. The test is applied to environmental samples already characterized by light microscopy counting. No significant difference is observed between the cell densities obtained by these two methods. This handy super-paramagnetic lateral flow immnunoassay biosensor can greatly assist water quality monitoring programs as well as ecological research.

Modelling and optimization of submicron Hall sensors for the detection of superparamagnetic beads

This paper deals with the numerical modeling of the electric potential distribution inside semiconductor Hall effect devices, under the presence of non-uniform magnetic fields. The model is applied to study miniaturized sensors for the detection of superparamagnetic nanobeads. The magnetic moment resolution is evaluated for different Hall probe geometries, by varying the bead size and its distance from the sensor surface.

Enzyme-linked immunosensor based on super paramagnetic nanobeads for easy and rapid detection of okadaic acid

Okadaic acid (OA), a lipophilic phycotoxin highly toxic to humans is produced by toxigenic dinoflagellates. The need to develop high performing methods for OA analysis able to improve the traditional ones is evident. In this work, competitive indirect enzyme-linked electrochemical immunosensor based on super paramagnetic nanobeads has been developed for the detection of OA. Streptavidin-coated magnetic beads were used as support to immobilize the biotinylated OA. Preliminary, colorimetric tests were performed in order to optimize different experimental parameters. Electrochemical detection was carried out by differential pulse voltammetry (DPV). The limit of detection (LOD) (0.38 μg L −1), the mid point value (IC 50) (3.15 μg L −1) and the time needed (60 min) for analysis of a real sample validated the developed electrochemical immunosensor as a promising tool for routine use. The matrix effect and the recovery rate were also assessed, showing an excellent percentage of recovery.

Single particle detection: Phase control in submicron Hall sensors

We present a phase-sensitive ac-dc Hall magnetometry method which allows a clear and reliable separation of real and parasitic magnetic signals of a very small magnitude. High-sensitivity semiconductor-based Hall crosses are generally accepted as a preferential solution for non-invasive detection of superparamagnetic nanobeads used in molecular biology, nanomedicine, and nanochemistry. However, detection of such small beads is often hindered by inductive pick-up and other spurious signals. The present work demonstrates an unambiguous experimental route for detection of small magnetic moments and provides a simple theoretical background for it. The reliability of the method has been tested for a variety of InSb Hall sensors in the range 600 nm–5 μm. Complete characterization of empty devices, involving Hall coefficients and noise measurements, has been performed and detection of a single FePt bead with diameter of 140 nm and magnetic moment of μ≈108 μB has been achieved with a 600 nm-wide sensor.

Nanoparticle-enabled highly sensitive detection of DNA methylation in single tube

Abstract Background: One of the often occurred epigenetic events associated to cancer is inheritable transcriptional silencing of the tumor suppressor genes by aberrant methylation of cytosines at the promoter regions. Current approaches to detect promoter DNA methylation commonly involve three separate independent processes, including DNA extraction, bisulfite conversion and methylation detection through PCR amplification, such as methylation specific PCR (MSP). This method includes many disconnected steps with loss of genetic materials, potentially reducing the sensitivity required for analysis of challenging clinical samples. Methods and Results: To address this problem, we developed a new technique named Methylation-on-Beads (MOB). MOB uses the silica superparamagnetic nanobeads (SSB) to combine DNA extraction, bisulfite treatment (Bst) and PCR into a single tube. The single-tube scheme minimizes the DNA loss during the tube transfers and improves the analytical sensitivity for methylation detection. Further enhancement in sensitivity is achieved by combining MOB with quantum dot-enhanced MSP detection. The pre-PCR DNA yields using MOB were compared with those carried out with conventional organic solvent extraction and ethanol precipitation (PC). 15 serum samples from lung cancer patients (7 Stage I, 3 Stage II, 5 Stage III) were analyzed using both MOB and PC. The DNA recovery using MOB was higher than PC for each patient serum sample with a median increase of 6.61 fold. To demonstrate the versatile applicability of the new method, we further analyzed 10 samples to include fresh tissue and paraffin embedded tissue from normal patients, fresh tumors from cancer patients and sputum samples. Median DNA yield increase of 7.8, 5.3, 6.4 and 7.5 respectively was determined using MOB when compared to the conventional method. We examined the outcome of bisulfite conversion in the presence of SSBs using real-time methylation specific PCR (MSP) to analyze p16INK4a promoter methylation. A set of triplicate reactions were examined with bisulfite-treated DNA of varying treatment durations (0h, 1h, 3h, 4h, 8h), and compared to the control using 16 hrs of conventional bisulfite treatment. Results indicated that 4h of bisulfite treatment was sufficient for conversion, and that the presence of beads did not alter the conversion process. Assessment of methylation in serum or plasma can be a useful tool for early detection of cancer. However, extending the DNA methylation analysis to clinically usable serum/ blood-based tests has been limited by the lack of sensitivity of conventional methods. In order to address whether improving DNA yields could enhance methylation detection, we compared methylation of p16INK4a promoter in 49 patient serum samples (18 normal and 31 cancer) in a blinded study using both MOB and PC/Bst/MSP. The 31 tumor samples were pre-selected from patients diagnosed with lung cancer who were also methylated for p16INK4a promoter in corresponding tumors. While p16INK4a methylation was detected in 14/31 patients with lung cancer using conventional approach; using MOB, we were able to detect p16INK4a methylation in 23/31of these patients. When samples used for methylation analysis contained large amounts of DNA (cell lines, tumors etc), a single-tube analysis of entire input amount may be unnecessary. Instead, multiple reactions in parallel were feasible by directly splitting the SSB into several different tubes. Conclusion: MOB successfully combined three processes required for DNA methylation analysis into a single-tube using SSB thereby allowing for ease in handling and increased throughput in detection. Increased pre-PCR yield in MOB allowed for efficient, diagnostically sensitive methylation detection.

Anti‐Clostridium tetani Antibody Determination in Serum Samples by Amperometric Immunosensing

AbstractAn electrochemical (EC) immunosensing assay for anti‐Clostridium tetani antibody determination in serum has been developed. The antigen tetanus toxoid was immobilized on superparamagnetic nanobeads. The immunoreaction occurred in Eppendorf minitubes. The anti‐tetani antibody was incubated in the presence of the toxoid functionalized nanobeads, then reacted with horseradish peroxidase‐labeled anti‐IgG. The resulting immunobeads were retained onto the carbon paste working electrode with a magnet. Hydroquinone served as redox label. The level of anti‐Clostridium tetani antibody in guinea pig serum samples was determined by amperometry using a carbon based screen‐printed electrode (cSPE) housed onto a magnetic support. The EC response was proportional to the logarithm of the antibody concentration comprised between 0.0046 IU/mL and 0.175 IU/mL with a limit of detection of 0.0046 IU/mL. In order to minimize the matrix effect, the standard addition method was applied. The assay was validated by comparing the EC immunosensing data with those obtained by applying the ELISA method described in the European Pharmacopoeia.

Detection of Magnetic Nanobeads by Self-Assembly of Superparamagnetic Microbeads for Biosensing

Detection of magnetically labeled biomolecules is a promising method for fast and inexpensive medical diagnosis. Recently, there has been an increasing interest in using magnetic labels with diameters of 20-150 nm-dimensions that are similar to actual biomolecules. However, detection of small numbers of nanometer-scale magnetic beads (labels) distributed over large surface areas by magnetoresistive devices is extremely challenging due to the intrinsic noise and design limitations of giant magnetoresistance (GMR) and Hall-type of biosensors. Here, we describe proof of principle experiments on exploiting magnetically induced self-assembly of superparamagnetic beads for detecting superparamagnetic nanobeads with diameters of less than 150 nm. We successfully detected low areal densities of ~ 130 nm diameter superparamagnetic ldquotarget beadsrdquo immobilized over millimeter-sized substrates, by optically monitoring the ldquocapturerdquo of easily visible, micrometer-sized superparamagnetic beads by the targets. Our procedure could readily be extended for the detection of magnetically labeled biomolecules.

Surface Plasmon Resonance Detection Using Antibody-Linked Magnetic Nanoparticles for Analyte Capture, Purification, Concentration, and Signal Amplification

Rapid, sensitive, and accurate detection of analytes present in low concentrations in complex matrixes is a critical challenge. One issue that affects many biosensor protocols is the number and nature of the interferences present in complex matrixes such as plasma, urine, stool, and environmental samples, resulting in loss of sensitivity and specificity. We have developed a method for rapid purification, concentration, and detection of target analytes from complex matrixes using antibody-coated superparamagnetic nanobeads (immunomagnetic beads, or IMBs). The surface plasmon resonance (SPR) detection signal from staphylococcal enterotoxin B (SEB) was dramatically increased when the IMBs were used as detection amplifiers. When SEB detection included a 10-fold concentration/purification IMB protocol, a substantial increase in detection sensitivity was observed. This procedure was used to successfully purify and concentrate SEB from serum and stool samples, then amplify the SPR detection signal. SEB at a concentration of 100 pg/mL was easily detected in both buffer and stool samples using this procedure. The IMB protocol also served to verify the analyte detection by using two different anti-SEB antibodies, mouse monoclonal antibodies attached to the magnetic nanobeads and rabbit polyclonal antibodies on the SPR sensor surface. Multiple detections of SEB in stool were performed using the same sensor surface by regenerating the sensor surfaces with a pH 2.2 buffer wash.

Development of lateral flow immunoassay system based on superparamagnetic nanobeads as labels for rapid quantitative detection of cardiac troponin I

In this study, superparamagnetic nanobeads (SPMNBs) were used as labels to establish a lateral flow immunoassay system for rapid, quantitative detection of cardiac troponin I (cTnI). First, the immunobeads were prepared by coupling monoclone antibody specific to cTnI onto SPMNBs. Then some factors which may influence the detection sensitivity of this lateral flow immunoassay system were studied, such as the amount of antibody immobilized in the Test line, sample volume and the amount of antibody per SPMNB. Finally, lateral flow test strips for detection of cTnI were established and applied to testing standard samples of different cTnI concentrations, which were also measured by Enzyme-linked Immunosorbent Assay (ELISA) in parallel. Furthermore, testing time and stability of magnetic signal were also investigated in this study. Results showed that this lateral flow test for cTnI had a high detection sensitivity of 0.01 ng/ml with a wide detection range of 5 orders of magnitude. The testing time was less than 15 min, and no significant decline of magnetic signal was observed in 140 days. Therefore, this lateral flow immunoassay system using SPMNB as labels will be a new point of care test (POCT) for rapid, quantitative detection of cardiac troponin I.

Preparation and application of surface-coated superparamagnetic nanobeads in the isolation of genomic DNA

A novel method was developed to prepare functionalized magnetic beads, in which superparamagnetic Fe3O4 core was synthesized with an injection-precipitation method and then was coated with functional groups using one-step suspension polymerization. In the coating and functionalizing process, a unique coupling reagent, bis-(2-hydroxyethyl methacrylate) phosphate, was introduced so that the monomers polymerized only on the surface of the nanocrystals without forming separate nuclei. The thickness of the coating layer and the size and density of the coated nanobeads were controlled by changing the quantity of the coated monomers. The nanobeads were characterized by transmission electron microscopy, light scattering spectrometry, Fourier transformation infrared spectroscopy, X-ray fluorescence spectroscopy and the magnetic hysteresis loop determination method. The carboxyl-modified magnetic nanobeads were employed to simplify the isolation of genomic DNA from human whole blood.

Preparation and application of surface-coated superparamagnetic nanobeads in the isolation of genomic DNA

A novel method was developed to prepare functionalized magnetic beads, in which superparamagnetic Fe 3O 4 core was synthesized with an injection-precipitation method and then was coated with functional groups using one-step suspension polymerization. In the coating and functionalizing process, a unique coupling reagent, bis-(2-hydroxyethyl methacrylate) phosphate, was introduced so that the monomers polymerized only on the surface of the nanocrystals without forming separate nuclei. The thickness of the coating layer and the size and density of the coated nanobeads were controlled by changing the quantity of the coated monomers. The nanobeads were characterized by transmission electron microscopy, light scattering spectrometry, Fourier transformation infrared spectroscopy, X-ray fluorescence spectroscopy and the magnetic hysteresis loop determination method. The carboxyl-modified magnetic nanobeads were employed to simplify the isolation of genomic DNA from human whole blood.